Modifided carboxypeptidase enzymes and their use

ABSTRACT

The invention relates to improvements relating to cancer therapy based on the identification of a number of regions of CPG2 which contain epitopes which appear to be involved in the production of a host immune response and which may be modified to alter the immunogenicity in patients. Production of fusions of CPG2 with an antibody, where the CPG2 protein has been tagged provides a CPG2 protein which has reduced immunogenicity. By using partially glycosylated enzyme obtainable by  P. pastoris  expression, the efficacy of antibody-CPG2 fusions is enhanced.

This application claims benefit of U.S. Provisional Application No.60/216,689, the entire contents of which is hereby incorporated byreference.

FIELD OF THE INVENTION

The present invention relates to improvements to the enzymecarboxypeptidase G2, and the use of this enzyme in therapy, particularlyantibody-directed enzyme prodrug therapy (ADEPT) and gene-directedenzyme prodrug therapy (GDEPT).

BACKGROUND TO THE INVENTION

Over the years, many cytotoxic compounds have been discovered which areof potential use in cancer chemotherapy. For example, nitrogen mustardsform one important family of such cytotoxic compounds. A problem withthe clinical use of cytotoxic compounds is in achieving sufficientselectivity in the cytotoxic effect between tumour cells and normalcells. One approach to address this problem has involved the developmentof so-called prodrugs which are derivatives of the cytotoxic drug, oftenrelatively simple derivatives, whose cytotoxic properties areconsiderably reduced compared to those of the parent drugs. Numerousproposals have been made for the administration of such prodrugs topatients under regimes whereby the prodrug is only converted by theaction of an enzyme to the cytotoxic drug in the region of the intendedsite of action.

A variety of systems exist for delivery of the enzyme. One such systemis described in WO88/07378, and involves conjugating the enzyme to anantibody specific for a tumour marker, delivering the antibody enzymeconjugate to a patient, allowing the conjugate to localise, and thendelivering the prodrug to the patient. This system is referred to asantibody-directed enzyme prodrug therapy” (ADEPT).

Another approach for delivery of the enzyme to the desired site ofaction is by the use of a genetic construct, such as a viral ornon-viral vector carrying a gene encoding the prodrug-converting enzyme,which is delivered to cells at the desired site of action (Huber et al,Proc. Natl. Acad. Sci. USA (1991) 88, 8039). A further alternativesystem is to provide a ligand, generally a naturally occurringpolypeptide whose biological role involves its binding to a cognatereceptor on the surface of the cell, conjugated to theprodrug-activating enzyme. This system, LIDEPT, is described inWO/97/26918, where VEGF is particularly exemplified as an example of aligand. A further alternative system is to use bacterial deliverysystems, for example, Clostridium or Salmonella based systems, in whichbacteria selectively colonise tumours. A Clostridium based system isdescribed in, for example, Fox et al, 1996 Gene Therapy 3 173-178.

One class of prodrugs suggested for use in the above systems is that ofprodrugs of nitrogen mustard compounds. Benzoic acid nitrogen mustardsare bifunctional alkylating agents, and a variety of prodrugs of suchcompounds are described in the art. One class of such prodrugs comprisea protecting group which may be removed by the action of acarboxypeptidase enzyme, such as bacterial carboxypeptidase G,particularly the Pseudomonas-derived enzyme carboxypeptidase G2 (CPG2).CPG2 is a well characterised enzyme with no mammalian equivalent. It isa non-covalently associated, homo-dimeric, metalloenzyme which cleavethe C-terminal glutamic acid of folate to yield a pteroate derivative.This has been exploited to cleave glutamic acid from a variety ofprodrugs to release potent nitrogen mustard compounds.

Examples of prodrugs which may be activated by CPG2 are described in,for example, Springer et al., Anti-Cancer Drug Design (1991) 6; 467-479,WO88/07378, WO94/25429 and WO96/22277. Fusions of an antibody fragmentdirected against carcinoembryonic antigen (CEA) with CPG2 have beendescribed in Michael et al, Immunotechnology 2 47-57 (1996) and the useof this fusion in in vivo model systems has been described in Bhatia etal, Int. J. Cancer, 85; 571-577 (2000).

A feature of CPG2 is that being a bacterial enzyme, it does not occurnaturally in the body of a human patient, and thus prodrugs designed tobe activated by this enzyme will not be activated elsewhere in thepatient. However, the drawback to this feature is that the enzymeprovokes an immune response in a patient, and indeed such responses havebeen observed in clinical trials of ADEPT using CPG2 (Sharma et al,1992, Cell Biophys., 21;109-120; Bagshawe et al, 1995, Tumour Targeting,1; 17-30).

DISCLOSURE OF THE INVENTION

We have investigated the immunogenicity of CPG2 and identified a numberof regions of this enzyme which contain epitopes which appear to beinvolved in the production of a host immune response. We have found thatwhere a host immune response is caused by the presence of such epitopes,these epitopes may be modified to alter the immunogenicity in patients.However the invention is not limited to this aspect alone, sincemodifications to these epitopes may be provided which render the CPG2less reactive with sera from CPG2 immunised patients. The latter aspectof the invention is also advantageous, to allow the development of CPG2fusions which “escape” or “evade”, to a greater or lesser degree, animmune response of a host which has been provoked by a wild-type CPG2 oranother altered form of CGP2 which has a set of one or more epitopemodifications which cause an established host response to a previouslyadministered form of CGP2 to be less effective against the newly alteredform.

Throughout this text where specific amino acids or amino acid sequencesof the Pseudomonas CPG2 used in the examples below are referred to wehave used the numbering used in the Swiss Prot database entry for CPG2(accession number p06621). The unprocessed form of CPG2 has a sequence415 amino acids long. The first 22 residues of this sequence are removedin the processed form of CPG2. In the MFE-23::CPG2 fusion proteindescribed here, the first amino acid of the CPG2 domain is amino acid 25according to the Swiss Prot CPG2 entry.

In a first aspect, the invention provides a CPG2 enzyme in which animmunogenic region selected from:

-   KIKGRGGK (amino acids 98-105, SEQ ID NO:1)-   KEYGVRD (157-163, SEQ ID NO:2), preferably YGVRD (159-163)-   KLADY (191-195, SEQ ID NO:3)-   GAGK (412-C-terminal(415), SEQ ID NO:4), preferably AG (413-414),-   EGGKKLVDK (331-338, SEQ ID NO:5)    is modified to reduce or alter immunogenicity to a mammalian immune    system whilst retaining CPG2 activity.

In another aspect, we have also found that production of fusions of CPG2with an antibody, where the CPG2 protein has been tagged provides a CPG2protein which has reduced immunogenicity. Thus in a further aspect ofthe invention, there is provided a CPG2 enzyme, including any of thoseof the first aspect, which is tagged with a his or myc-his tag.

There is also provided by the invention methods of treatment ordiagnosis by methods such as ADEPT, GDEPT or LIDEPT which utilise theCPG2 of the present invention. These and other aspects of the inventionare described herein in more detail below.

DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates inhibition of CPG2 binding of human antibodies byCM79 determined by ELISA. Sera A1 to A10 were from ten patients posttreatment with an A5B7-CPG2 antibody-enzyme conjugate used in an ADEPTclinical trial. Values are mean±SEM.

FIG. 2 illustrates three segments of the SELDI epitope mapping massspectra of CPG2 with CM79 (left hand spectra) and with non-CPG2 bindingsFv M009 controls (right hand spectra).

2A) Glu-C digested CPG2 peptides R[356-415]K (mass 6269.8 Da) andS[376-415]K (mass 4354.1 Da).

2B) Glu-C digested CPG2 peptides Y[391-415]K (mass 2794.9) andY[159-189]E (mass 3539.9 Da).

2C) Glu-C digested CPG2 peptides Y[159-176]E (mass 2092.8 Da)

FIG. 3A shows a representation of the X-ray crystal derived structure ofCPG2, showing regions I and II.

FIG. 3B shows the amino acid sequence of CPG2.

FIG. 4 illustrates CM 79 binding to wt CPG2, variant V6 and variant V8,determined by ELISA.

FIG. 5 illustrates an example of patient serum (A21) binding to wt CPG2and variant V6. Binding was determined by ELISA. (Serum dilution 0.1=a 1in 10 fold dilution.

FIG. 6 illustrates plasma clearance of CPG2 activity in LS174Txenografted nude mice given MFE23::CPG-gly-his fusion protein.

FIG. 7 illustrates biodistribution of CPG2 activity in LS174Txenografted nude mice given MFE23::CPG-gly-his fusion protein.

FIG. 8 illustrates tumour to normal tissue ratios in LS174T xenograftednude mice given MFE23::CPG-gly-his fusion protein.

FIG. 9A illustrates time-activity curves for actual tumour and blooddata for the glycosylated fusion protein along with the models thatdescribe blood clearance and uptake of the non-specific (NS) antibody intumour.

FIG. 9B illustrates time-activity curves for actual tumour and blooddata for the non-glycosylated fusion protein along with the models thatdescribe blood clearance and uptake of the non-specific (NS) antibody intumour.

FIG 10A illustrates the effect of ADEPT therapy in LS174T xenograftednude mice given MFE23::CPG-gly-myc-his fusion protein in combinationwith bis-iodo phenol mustard prodrug (ZD2767P).

FIG 10B illustrates the effect of ADEPT therapy in LS174T xenograftednude mice given MFE23::CPG-gly-his fusion protein in combination withbis-iodo phenol mustard prodrug (ZD2767P).

FIG. 11A illustrates toxicity measured as weight loss in LS174Txenografted nude mice given MFE23::CPG-gly-myc-his fusion protein incombination with bis-iodo prodrug.

FIG. 11B illustrates toxicity measured as weight loss in LS174Txenografted nude mice given MFE23::CPG-gly-his fusion protein incombination with bis-iodo prodrug.

FIG. 12 illustrates the CPG2 molecule showing the molecular dynamicspredicted interaction of a charged histidine residue in the hexa-His-tagwith a residue from the epitope KEYGVRD, residues 157-163.

FIG. 13 illustrates the CPG2 molecule (chain A only) showing themolecular dynamics predicted interaction of an uncharged histidineresidue in the hexa-His-tag with a residue from the epitope EGGKKLVDK,residues 331-339, the prediction assuming an uncharged His-tag.

FIG. 14 illustrates the CPG2 molecule (chain A only) showing themolecular dynamics predicted interaction of a glutamate residue in theMyc tag with a residue from the epitope EGGKKLVDK, residues 331-339.

DETAILED DESCRIPTION OF THE INVENTION

The modifications to the immunogenic regions identified above may be anytype of modifications which have the defined function of retaining CPG2activity and being reduced or altered in immunogenicity. These areessentially functional tests which those of skill in the art can performusing routine skill and knowledge.

Thus for example, CPG2 activity may be tested by the ability of theenzyme to hydrolyse methotrexate (MTX). CPG2 hydrolysis of MTX resultsin a change in absorbance at 320 nm which may be measured byspectrophotometry. CPG2 catalytic activity in solid tissues can beassayed using an indirect HPLC method, measuring, for example,2,4-diamino-N¹⁰ methypteroic acid (DAMPA, a metabolite of MTX). Examplesof further tests are described in McCulloch et al, J. Biol. Chem.246:7207-7213, 1971 and Sherwood et al, Eur. J. Biochem. 148: 447-453,1985.

The reduction or alteration in immunogenicity of the modified enzyme maybe determined by injecting the enzyme into a test animal, typically amouse, and assaying the immune response of the mouse to one or more—e.g.2, 3, 4 or 5 repeat injections of the modified enzyme compared to anunmodified control. Such a protocol is exemplified in the accompanyingexamples, and may be used in similar form.

Particular modifications include:

-   -   substitutions    -   deletions    -   insertions    -   replacement of the immunogenic regions by human sequences of        sequence in particular at positions where intramolecular        interactions are observed to be present in the X ray structure.

These replacements may be made to retain the correct stereochemistry,hydrophobicity or charge characteristics, even where sequence similaritymay be low.

Examples of substitutions include the replacement of charged residuesfor uncharged residues, uncharged residues for charged residues, polarresidues for non-polar residues, non-polar residues for polar residues,large side chain residues for smaller side chain residues, small sidechain residues for larger side chain residues. Specific residues thatmay be substituted include R162 and G412. Other CPG2 amino acids to besubstituted will be identified using anti-CPG2 antibodies/antibodyfragments particularly where these can block human anti-CPG2 antibodiesas identified using enzyme linked immunosorbent assay (ELISA). Theseanti-CPG2 antibodies will be epitope mapped. Subsequent substitution ofamino acids in these CPG2 epitopes will be of those type mentioned.

The MFE-23::CPG2-his fusion protein has a hexa-His tag at the C-terminusof CPG2. This tag may be extended by inserting sequences of varyinglength between the hexa-His tag and the C-terminus of CPG2. Insertionsinclude the myc tag (EQKLISEEDLN) to result in a myc-his tag having thesequence AAASFLEQKLISEEDLNSAVDHHHHHH, or a humanized version of the myctag. Such insertions may serve to mask immunogenic surfaces on the CPG2protein.

Usually, no more than 10, for example from 5 to 10, such as 5,preferably no more than 4, for example 3, 2 or just one substitutionwill be made to the native CPG2 sequence in each of the immunogenicregions identified. The enzyme may comprise 1, 2, 3, 4 or from 5 to 10substituted immunogenic regions.

Similarly, no more than 10, for example from 5 to 10, such as 5,preferably no more than 4, for example 3, 2 or just one deletion will bemade to the native CPG2 sequence in each of the immunogenic regionsidentified. The enzyme may comprise 1, 2, 3, 4 or from 5 to 10immunogenic regions carrying a deletion.

Likewise, no more than 10, for example from 5 to 10, such as 5,preferably no more than 4, for example 3, 2 or just one insertion willbe made to the native CPG2 sequence in each of the immunogenic regionsidentified. The enzyme may comprise 1, 2, 3, 4 or from 5 to 10immunogenic regions in which an insertion has been made. Examples ofinsertions include the replacement of surface loops, extensions ofeither the C-terminus or the N-terminus or the replacement of anysegmented sequence where the exposed residues may be substituted forother residues but where the buried residues are conserved so as tominimise disruption of the 3-D structure.

In the case of the C-terminal immunogenic region, the modification maybe by way of an extension to the C-terminus of the enzyme. We havesurprisingly found that addition of a polyhistidine tag to theC-terminus of CPG2 provided a significant reduction in immunogenicity.The further addition of a myc tag to the polyhistidine tag provided afurther decrease.

The histidine tag is a synthetic tag widely used in the art to aidprotein purification or identification. It is not a native human ormurine epitope and it is therefore surprising that its addition isbeneficial in reducing the immune response. The myc tag is derived fromthe c-myc proto-oncogene and would normally not be expected to provoke aresponse in its native conformation in humans.

C-terminal extensions may thus be selected broadly, although in generalterms such extensions will typically be short peptide sequences of, forexample, from 5 to 20 amino acids, and may be synthetic sequences ornatural sequences of mammalian, particularly human origin. Two or more,such as from 2 to 5 such sequences (which may be the same or different)may be added in tandem.

Replacement of these immunogenic regions may be by human sequences ofsimilar sequence to the wild type sequence, or by sequences exhibitingsimilar conformations, or by sequences exhibiting similar hydrophobic,charge, stereochemical or surface exposure characteristics. In anexample procedure, the sequence, conformation and interior proteincontact profile of an immunogenic region would be encoded as a set ofcriteria on which to search and select similar regions from a databaseof human protein three-dimensional structures (for example, the proteindatabank (PDB) could be searched using the IDITIS software (OxfordMolecular plc, UK)). Such selected regions would then be ranked on theirsimilarity to the above criteria and the most similar sequence orsequences used for replacement of an existing immunogenic region. Wherethe humanised sequences comprise the same or similar residues involvedin internal packing interactions as the wild type sequence, thehumanised sequence may not require further modification. If, however,these residues result in structural perturbations of the internalinteractions with the rest of the molecule, these buried residues may besubstituted with residues present in the wild-type molecule, producing ahybrid sequence which retains internal packing interactions andstereochemistry. Examples of suitable regions found using this methodfor replacement of the KEYGVRD sequence (SEQ ID NO:2) include the hybridsequence (maintaining intramolecular contacts) YEYGVMK of the humanisedsequence YEVGMMK. Examples of suitable sequences for replacement ofKLADY (SEQ ID NO:3) include the hybrid sequence (maintainingintramolecular contacts) RNSDY of the humanised sequence RNSDR.

In a related aspect of the invention, we have found that expression ofMFECPG2 in Pichia pastoris provides advantages over bacterially producedCPG2. The enzyme CPG2 is bacterial and thus in its native form is notglycosylated. However, the sequence of the enzyme contains three motifsAsn-Xaa-Thr/Ser which are recognised by eukaryotic cells as targets forN-linked glycosylation, (1), (2) and (3). These three Asn residues whichare glycosylated are found at positions 222, 264 and 272 of PseudomonasCPG2. As mentioned above, CPG2 is a homo-dimer and it has been foundthat N-linked glycosylation appears to interfere with the formation ofthe dimer, particularly at its second position, 264.

Surprisingly, we have found that production of the enzyme in P. pastorisresults in glycosylation only at residues 222 and 272, thus reducing theinterference caused by glycosylation at 264 which occurs in CPG2produced in, for example, mammalian cells.

We have also found that by using partially glycosylated enzymeobtainable by P. pastoris expression, the efficacy of antibody-CPG2fusions is enhanced, in that the enzyme localises to the tumour whilstbeing cleared efficiently from other organs and the bloodstream of themammalian body.

Thus the invention provides a CPG2 enzyme which is partially N-linkedglycosylated at one or both of positions (1) and (3), and not N-linkedglycosylated at position (2). Preferably, position (2) retains itsnative sequence and is not glycosylated as a result of expression in P.pastoris.

Expression of the CPG2 wherein the motifs (1), (2) and (3) are allpresent is preferably in a P. pastoris host cell, or a host cell of ayeast, such as a methylotrophic yeast. Such yeasts are those which arecapable of growth on methanol and include yeast of the genera Hansenula,Candida, Kloeckera, Pichia, Saccharomyces, Torulopsis and Rhodotorula. Alist of specific species which are exemplary of this class of yeasts maybe found in C. Anthony, The Biochemistry of Methylotrophs, 269 (1992).Pichia, particularly P.pastoris, is preferred.

In the present invention, a preferred CPG2 enzyme which is used is theCPG2 whose sequence is disclosed in WO88/07378 and herein in FIG. 3B,the disclosure of which is incorporated herein by reference. However,other bacterial carboxypeptidase enzymes may be used, e.g., CPG2 enzymesfrom Variovorax species such as Variovorax paradoxes and CPG2 enzymesfrom other Pseudomonas species such as Pseudomonas aeruginosa,Pseudomonas cepacia, Pseudomonas fluorescens, Pseudomonas putida,Pseudomonas syringae, Pseudomonas savastanoi, which in the native formcomprise three asparagine residues, Asn (1), Asn (2), Asn (3) numberedin the N-terminal to C-terminal direction, the residues being part ofmotifs which on expression in a mammalian cell are subject to N-linkedglycosylation. In such enzymes Asn (1), Asn (2) and Asn(3) will be atpositions homologous to Asn 222, Asn 264 and Asn 272, although they mayhave different positional numbering. However, Asn (1), Asn (2) and Asn(3) of these enzymes can readily be identified by persons skilled in theart, for example using sequence alignments to compare a sequence withthe sequence shown herein, and thereby identify the Asn residues whichcorrespond to Asn 222, Asn 264 and Asn 272 of FIG. 3B. Likewise, theepitopes identified as SEQ ID NOS: 1 to 5 above may have differentpositional numbering and/or minor modifications to the wild typesequence but can be determined by those skilled in the art by themodelling techniques described herein by analogy to FIG. 3B. CPG2enzymes from other species of Pseudomonas may be obtained by routinecloning methodology. For example, a library of CDNA from a Pseudomonasspecies may be made and probed with all or a portion of the sequence ofFIG. 3B under conditions of medium to high stringency.

For example, hybridizations may be performed, according to the method ofSambrook et al. (below) using a hybridization solution comprising: 5×SSC(wherein ‘SSC’=0.15 M sodium chloride; 0.15 M sodium citrate; pH 7), 5×Denhardt's reagent, 0.5-1.0% SDS, 100 μg/ml denatured, fragmented salmonsperm DNA, 0.05% sodium pyrophosphate and up to 50% formamide.Hybridization is carried out at 37-42 C for at least six hours.Following hybridization, filters are washed as follows: (1) 5 minutes atroom temperature in 2×SSC and 1% SDS; (2) 15 minutes at room temperaturein 2×SSC and 0.1% SDS; (3) 30 minutes-1 hour at 37 C in 1×SSC and 1%SDS; (4) 2 hours at 42-65 C in 1×SSC and 1% SDS, changing the solutionevery 30 minutes.

Clones identified as positive may be examined to identify open readingframes encoding homologues of the sequence shown in FIG. 3B. It may benecessary to combine more than one clone to achieve a full length openreading frame, as would be understood by the person skilled in art.Clones may then be expressed in a heterologous expression system, e.g.in bacteria or yeast and the protein purified by techniques known in theart.

Alternatively, CPG2 enzyme producing bacteria may be identified bymethods involving the identification of organisms that convert folicacid to pteroate or of organisms capable of growing on media with folicacid as the sole carbon source.

Suitable enzymes to which mutations according to the invention may beapplied include carboxypeptidase enzymes which are mutants, variants,derivatives or alleles of the sequence shown in FIG. 3B. Acarboxypeptidase enzyme which is a variant, allele, derivative or mutantmay have an amino acid sequence which differs from that given in FIG. 3Bby one or more of addition, substitution, deletion and insertion of oneor more amino acids, for example from 1 to 20, such as from 1 to 10,e.g., 1,2,3,4,5 or 6-10 substitutions deletions or insertions.

Preferred such carboxypeptidases will have the ability to hydrolysemethotrexate (MTX). Alteration of sequence may change the nature and/orlevel of activity and/or stability of the carboxypeptidase enzyme.

A polypeptide which is an amino acid sequence variant, allele,derivative or mutant of the amino acid sequence shown in FIG. 3B maycomprise an amino acid sequence which shares greater than about 35%sequence identity with the sequence shown, greater than about 40%,greater than about 50%, greater than about 60%, greater than about 70%,greater than about 80%, greater than about 90% or greater than about95%. The sequence may share greater than about 60% similarity, greaterthan about 70% similarity, greater than about 80% similarity or greaterthan about 90% similarity with the amino acid sequence shown in FIG. 3B.Amino acid similarity is generally defined with reference to thealgorithm GAP (Genetics Computer Group, Madison, Wis.) as noted above,or the TBLASTN program, of Altschul et al. (1990) J. Mol. Biol. 215:403-10. Parameters employed are the default ones: for nucleotidesequences—Gap Weight 50, Length Weight 3, Average Match 10.000, AverageMismatch 0.000; for peptide sequences—Gap Weight 8, Length Weight 2,Average Match 2.912, Average Mismatch −2.003. Peptide similarity scoresare taken from the BLOSUM62 matrix. Also useful is the TBLASTN program,of Altschul et al. (1990) J. Mol. Biol. 215: 403-10, or BestFit, whichis part of the Wisconsin Package, Version 8, September 1994, (GeneticsComputer Group, 575 Science Drive, Madison, Wis., USA, Wisconsin 53711).Sequence comparisons may be made using FASTA and FASTP (see Pearson &Lipman, 1988. Methods in Enzymology 183: 63-98). Parameters arepreferably set, using the default matrix, as follows: Gapopen (penaltyfor the first residue in a gap): −12 for proteins/−16 for DNA; Gapext(penalty for additional residues in a gap): −2 for proteins/−4 for DNA;KTUP word length: 2 for proteins/6 for DNA.

Sequence comparison may be made over the full-length of the relevantsequence shown herein, or may more preferably be over a contiguoussequence of about or greater than about 20, 25, 30, 33, 40, 50, 67, 133,167, 200, 233, 267, 300, 333, or more amino acids or nucleotidetriplets, compared with the relevant amino acid sequence or nucleotidesequence as the case may be.

In further aspects the invention provides a nucleic acid encoding suchmodified bacterial carboxypeptidases or vectors comprising such nucleicacid. The vector is preferably an expression vector, wherein saidnucleic acid is operably linked to a promoter compatible with a hostcell. The invention thus also provides a host cell which contains anexpression vector of the invention.

Host cells of the invention may be used in a method of making acarboxypeptidase enzyme of the invention as defined above whichcomprises culturing the host cell under conditions in which said enzymeor fragment thereof is expressed, and recovering the enzyme insubstantially isolated form. The enzyme may be expressed as a fusionprotein.

Host cells may be used to provide fusions of antibody-enzyme conjugatesfor use in ADEPT therapy, for ligand-enzyme conjugates for LIDEPTtherapy, or may be used in the provision of vectors such as viralvectors for GDEPT therapies. ADEPT therapy has utility in the treatmentof tumours which are associated with tumour specific markers, which maybe the target for an antibody. By “antibody”, this is intended to referto any binding fragment thereof, characterised by the presence of a VH,and preferably also a VL region. Such fragments include single chain Fvand Fab fragments.

Examples of tumour antigens include CEA, a cell surface glycoproteinstrongly expressed by most colorectal tumours. Colorectal cancer is thesecond leading cause of cancer death in the UK. Conventional treatmentsremain unable to cure patients with advanced or metastatic disease. CEAis a suitable target for ADEPT because, with highly specific antibodies,CEA is only detectable on tumours and on the luminal surface of the gutwhich is not readily accessible to IgG antibodies. A particular anti-CEAantibody is MFE-23 which is disclosed in WO95/15341. Other antibodychemical conjugates with CPG2 which have been proposed for ADEPT therapyare the anti-human chorionic gonadotropin monoclonal antibody (MAb) W14F(ab′)2 and the anti-c-erbB2 MAb ICR12 (Bagshawe, 1998, Tumor targeting,3: 21-24, 1998) and the CPG2 of the present invention may be used insuch conjugates.

More generally however, for ADEPT it is not essential that the targetantigen is attached to the cell surface, as prodrug can be converted toactive drug in the tumour interstitial space and diffuse into the tumourcells. Targets that are secreted or cleaved from tumour cells, orproduced in tumour stroma, may be applicable. A target which isheterogeneously expressed, as often occurs with tumour antigens, issimilarly acceptable.

Thus other targets which exist include for example the idiotype inlymphomas or mutant cell-surface proteins, which are increasingly beingidentified, in other tumours [Urban JL and Schreiber H, (1992) Tumourantigens. Ann Rev Immunol 10, 617-644.] relative abundance may giveadequate selectivity for ADEPT although the normal tissue reactivityshould be well defined. In some cases the forms expressed in normaltissue may be relatively inaccessible to antibody, as for example withCEA discussed above.

Another target which has been investigated is p185^(HER2) which isupregulated in breast cancer. This antigen is also expressed on avariety of normal epithelial cells but in vitro experiments have shownthat the relative abundance of p185^(HER2) on a breast tumour cell linewas sufficient to allow specific targeting in ADEPT [Rodrigues M L, etal, (1995) Cancer Res. 55, 63-70]. Where antigens are expressed innormal tissues their pattern of distribution should be considered whenchoosing a suitable toxic agent. For example, the enzyme β-lactamase,genetically fused to anti-p185^(HER2), has been used to generate thedrug doxorubicin for which heart tissue is a site of chronic and doselimiting toxicity and bone marrow is a site of acute toxicity. As thereis no detectable expression of p185^(HER2) in heart or bone marrow theabove ADEPT combination seems particularly suitable for this antigen.

Tumour vasculature is an attractive target for some antibody therapiesas it is readily accessible and essential for tumour growth. Moreover,experimental models have demonstrated the potential efficacy of targetedimmunotoxins to kill tumour endothelial cells [Burrows F J and Thorpe PE, (1993) Proc Natl Acad Sci USA 90, 8996-9000]. Tumour vasculature mayprovide a good target for ADEPT if the prodrug is designed to have avery short half life so that active drug does not leak back into normaltissues via the blood.

Suitable viral vectors for VDEPT include those which are based upon aretrovirus. For GDEPT, a wide variety of vectors are available. Theseinclude those which are based upon a retrovirus. Such vectors are widelyavailable in the art. Huber et al (ibid) report the use of amphotropicretroviruses for the transformation of hepatoma, breast, colon or skincells. Culver et al (Science (1992) 256; 1550-1552) also describe theuse of retroviral vectors in GDEPT. Such vectors or vectors derived fromsuch vectors may also be used. Other retroviruses may also be used tomake vectors suitable for use in the present invention. Suchretroviruses include rous sarcoma virus (RSV). The promoters from suchviruses may be used in vectors in a manner analogous to that describedabove for MLV.

EP-A-415 731 describes molecular chimeras comprising a promoter whichmay be activated in a tumour cell operably linked to a heterologous geneencoding an enzyme capable of converting a prodrug into a cytotoxicagent. Such molecular chimeras may be used to express enzymes of theinvention in tumour cells in order to activate prodrugs. EP-A-415 731describes incorporation of such molecular chimeras into viral vectors,e.g. adenoviral or retroviral vectors. Such viral vectors may also beadapted for utilization in the present invention.

Other recombinant viral vector delivery systems are described inWO91/02805, WO92/14829, WO93/10814, WO94/21792, WO95/07994, WO95/14091and WO96/22277, the disclosures of which are incorporated herein byreference. Methods for producing vector delivery systems based on theabove-mentioned disclosures may be used to deliver vectors encoding theactivating enzyme to target cells.

Englehardt et al (Nature Genetics (1993) 4; 27-34) describes the use ofadenovirus based vectors in the delivery of the cystic fibrosistransmembrane conductance product (CFTR) into cells, and such adenovirusbased vectors may also be used in accordance with the present invention.Vectors utilising adenovirus promoter and other control sequences may beof use in delivering a system according to the invention to cells in thelung, and hence useful in treating lung tumours.

Vectors encoding the CPG2 carboxypeptidase may be made using recombinantDNA techniques known per se in the art. The sequences encoding theenzyme may be constructed by splicing synthetic or recombinant nucleicacid sequences together, or modifying existing sequences by techniquessuch as site directed mutagenesis. Reference may be made to “MolecularCloning” by Sambrook et al (1989, Cold Spring Harbor) for discussion ofstandard recombinant DNA techniques. In general, the vector may be anyDNA or RNA vector used in GDEPT therapies.

The CPG2 carboxypeptidase will be expressed from the vector using apromoter capable of being expressed in the cell to which the vector istargeted. The promoter will be operably linked to the sequences encodingthe enzyme and its associated sequences.

Suitable promoters include viral promoters such as mammalian retrovirusor DNA virus promoters, e.g. MLV, CMV, RSV and adenovirus promoters.Preferred adenovirus promoters are the adenovirus early gene promoters.Strong mammalian promoters may also be suitable. An example of such apromoter is the EF-1α promoter which may be obtained by reference toMizushima and Nagata ((1990), Nucl. Acids Res. 18; 5322). Variants ofsuch promoters retaining substantially similar transcriptionalactivities may also be used.

The c-erbB2 proto-oncogene is expressed in breast tissues at low levelsand in a tissue restricted manner. In some tumour states, however, theexpression of this protein in increased, due to enhanced transcriptionalactivity. Notable examples of this are breast tissue (about 30% oftumours), ovarian (about 20%) and pancreatic tumours (about 50-75%). Insuch tumours where expression of c-erbB2 is increased due to enhancedtranscription or translation, the c-erbB2 promoter may be used to directexpression of the activating enzyme in a cell specific manner. Thespecificity of GDEPT may be increased since transfection of normal cellsby a vector with a c-erbB2 promoter will provide only very limitedamount of enzyme or none and thus limited activation of prodrug. The useof the c-erbB2 promoter and homologous promoters in GDEPT is more fullydescribed in WO96/03151.

The prodrug for use in the system will be selected to be compatible withthe CPG2 carboxypeptidase such that the enzyme will be capable ofconverting the prodrug to the active drug. Desirably, the toxicity ofthe prodrug to the patient being treated will be at least one order ofmagnitude less toxic to the patient than the active drug. Preferably theactive drug will be several, e.g. 2, 3 or 4 or more orders of magnitudemore toxic than the prodrug. Nitrogen mustard prodrugs are preferred.Other suitable prodrugs include those disclosed in WO96/03515.

Nitrogen mustard prodrugs include compounds of the formula:M-Ar—CONH—Rwhere Ar represents an optionally substituted aromatic ring system, R—NHis the residue of an α-amino acid R—NH₂ or oligopeptide R—NH₂ andcontains at lease one carboxylic acid group, and M represents a nitrogenmustard group. The residue of the amino acid R—NH is preferably theresidue of glutamic acid. It is disclosed in WO88/07378 that the enzymecarboxypeptidase G2 is capable of removing the glutamic acid moiety fromcompounds of the type shown above, and the removal of the glutamic acidmoiety results in the production of an active nitrogen mustard drug.Prodrugs of a similar structure are also disclosed in WO94/02450, thedisclosure of which is incorporated herein by reference.

In a further aspect, the present invention provides a pharmaceuticalcomposition, medicament, drug or other composition comprising an enzymeof the invention. The composition may include a pharmaceuticallyacceptable carrier or diluent.

The invention also provides a kit comprising:

-   -   (a) a prodrug which can be converted to a cytotoxic drug by        CPG2; and one of    -   (b(i)) an immunoglobulin/enzyme fusion protein or conjugate in        which the immunoglobulin is specific for a cellular (e.g.        tumour-associated) antigen and the enzyme is a carboxypeptidase        enzyme;    -   (b(ii)) a ligand-enzyme conjugate or fusion protein, the ligand        being specific for a cellular (e.g. tumour associated antigen)        and the enzyme is a carboxypeptidase enzyme;    -   (b(iii)) a vector which encodes a carboxypeptidase enzyme which        can be expressed in a cell (e.g. tumour cell)    -   the carboxypeptidase being a carboxypeptidase of the invention.

In the kits of the invention, the vectors conjugates or fusion proteinsmay themselves be provided in a composition including a pharmaceuticallyacceptable carrier or diluent.

Compositions according to the present invention, and for use inaccordance with the present invention, may include, in addition toactive ingredient, a pharmaceutically acceptable excipient, carrier,buffer, stabiliser or other materials well known to those skilled in theart. Such materials should be non-toxic and should not interfere withthe efficacy of the active ingredient. The precise mature of the carrieror other material will depend on the route of administration, which maybe oral, or by injection, e.g. cutaneous, subcutaneous or intravenous.

Administration of the prodrug and/or vector and/or fusion and/orconjugate is preferably in a “therapeutically effective amount”, thatbeing sufficient to show benefit to the patient. The doses of eachcomponent and the route and time-course of their administration willultimately be at the discretion of the physician, who will take intoaccount such factors as the nature and severity of what is being treatedand the age, weight and condition of the patient.

Suitable doses of prodrug and conjugate for the ADEPT approach are givenin Bagshawe et al. Antibody, Immunoconjugates, and Radiopharmaceuticals(1991), 4, 915-922. A suitable dose of conjugate may be from 2000 to200,000 enzyme units/m² (e.g. 20,000 enzyme units/m²) and a suitabledose of prodrug may be from 20 to 2000 mg/m² (e.g. 200 mg/m²).

In order to secure maximum concentration of the fusion protein orconjugate at the site of desired treatment, it is normally desirable tospace apart administration of the two components by at least 4 hours.The exact regime will be influenced by various factors including thenature of the tumour to be targeted and the exact nature of the prodrug.A typical regime is to administer the conjugate at 0 h, galactosylatedclearing antibody at 24 h, and prodrug at 48 h. If no clearing antibodyis used, it would generally be longer than 48 h before the prodrug couldbe injected.

In using the LIDEPT systems of the present invention the prodrug willusually be administered following administration of the ligand-enzymefusion protein or conjugate. Typically, the ligand/enzyme will beadministered to the patient, and its uptake monitored, for example byrecovery and analysis of a biopsy sample of targeted tissue or byinjecting trace-labelled protein ligand enzyme.

In using the GDEPT system the prodrug may be administered followingadministration of the vector encoding the activating enzyme. Typically,the vector will be administered to the patient and then the uptake ofthe vector by transfected or infected (in the case of viral vectors)cells monitored, for example by recovery and analysis of a biopsy sampleof targeted tissue.

The amount of vector delivered will be such as to provide an effectivecellular concentration of enzyme so that the prodrug may be activated insufficient concentration at the site of a tumour to achieve atherapeutic effect, e.g. reduction in the tumour size. This may bedetermined by clinical trials which involve administering a range oftrial doses to a patient and measuring the degree of infection ortransfection of a target cell or tumour. The amount of prodrug requiredwill be similar to or greater than that for ADEPT systems of the typementioned above.

A treatment according to the present invention may be administered aloneor in combination with other treatments, either simultaneously orsequentially dependent upon the condition to be treated.

In one aspect, the finding of immunogenic “hot-spots” provides a noveltherapeutic method wherein a patient is administered different forms ofCPG2 of the invention in successive rounds of therapy, such that anyantibody response to a first modified CPG2 of the invention is notprovoked by a second, different CPG2 of the invention which isadministered in a second (or subsequent) round of therapy. Furthermore,a patient may be administered a wild-type form of CPG2 of the inventionin a first round of therapy, followed after the first round (which maybe more than one dose of wild-type CPG2) with a CPG2 of the presentinvention.

EXAMPLES OF THE INVENTION Example 1 Identification and Modification ofImmunogenic Epitopes

As described below, from a filamentous phage library of antibody genesobtained from CPG2 immunized mice we isolated two single chain Fvantibody fragments (CM79 and CM12) that partially blocked the polyclonalantibody response generated in patients who received CPG2 in ADEPTtherapy. Using specialized metal chips coated with CM79/CM12 followed byantigen binding, selective proteolysis and surface enhanced laserdesorption ionization affinity mass spectrometry (SELDI-AMS) theimmunogenic region of CPG2 was characterised as incorporating theC-terminus and three loops from sequentially remote sequences whichbound to both CM79 (C-terminus and one loop) and CM12 (three loops).(From these results, the immunogenic regions corresponding to SEQ IDNOS: 1,2,3,4 were derived. We confirmed and silenced the epitopescorresponding to SEQ ID NO:2 and SEQ ID NO:4 by mutagenesis to give CPG2variants with negligible binding to CM79. The variants showedsignificant reduction in reactivity against sera from patients withpost-therapy immune responses to wild-type CPG2.

1.1 Characterization of the Anti-CPG2 Antibody Phage Library.

An anti-CPG2 antibody library was generated containing 1.78×10⁷ sFvclones. The twelve sFv clones which gave the highest optical density(OD) readings in ELISA with CPG-coated plates were chosen. These werethen tested by serum inhibition ELISA for their ability to block thehuman polyclonal antibody response made by 2 patients who had receivedwild-type CPG2 in ADEPT clinical trials. Results showed a range of0%-20% inhibition of patients' sera binding by the sFvs. The strongestinhibitors, clones CM79 and CM12, were sub-cloned and purified to allowdetailed analysis. CM79-saturated CPG2 was presented to sera from 10CPG2-immunized patients and reductions of up to 18% of the IgG responsewere observed (FIG. 1). Although the examples below describe in detailthe procedures related to CM79 used to identify epitopes SEQ ID NO:2 andSEQ ID NO:4, it should be understood that similar procedures werecarried out starting with clone CM12 resulting in identification ofepitopes SEQ ID NO:1 and SEQ ID NO:3. The epitope corresponding to SEQID NO:5 was identified by molecular dynamics modelling as describedbelow.

1.2 Epitope Identification

Epitopes were identified using SELDI-AMS epitope mapping and predictionof surface exposed regions.

i) SELDI-AMS

CM79 was subcloned into a pUC119 hexahistidine-tag vector, expressed andIMAC purified (Casey, J. L. et al. J. Immunol. Methods 179, 105-116(1995)). CM79 mass was determined using SELDI-AMS. To an NP1 SELDI chip(Ciphergen Biosystems Plc, Ca., USA) 1 ml of CM79 (0.5 mg/ml indistilled water) was added with sinapinic acid matrix (5 mg/ml in 50%acetonitrile, 0.5% trifluoracetic acid). A PBS-1 mass spectrometer(Ciphergen Biosystems Plc.) was used to collect mass data. The laserintensity was 20 with 100 shots collected and averaged per sample. ForSELDI-AMS epitope mapping, all incubation stages were 1 h, roomtemperature, in a humidity chamber unless stated otherwise. 2 ml ofCM79-His or anti-CEA sFv M009-His (both 0.3 mg/ml), were incubated on aPS1 SELDI chip (Ciphergen Biosystems Plc.). 4 ml of 1 M ethanolamine (pH8) was added to each spot and incubated for 20 min. The chip was washedwith 4×5 μl of PBS+0.1% Triton X-100 (PBST) and submerged in PBST for 15min.

Antigens (1 ml of 3 mM CPG2+10 mM BSA, or 10 mM BSA) were incubated onthe chip. The chips were washed and incubated with Glu-C protease(Boehringer-Mannheim-Roche, UK). Enzyme to substrate ratios were 1:20 or1:50 diluted in PBST. Protease digestions were performed for 1.5 h.Matrix was 0.5 μl α-cyano-4-hydroxycinnamic acid (CHCA) at 5 mg/ml in50% acetonitrile/0.1% trifluoroacetic acid. Mass data was acquired usingthe PBS-1 mass spectrometer. 100 samples per spot with laser intensitysetting at 10 were collected and averaged in automatic mode. Calibrationwas external using bovine ubiquitin (8564.8 Da).

ii) Predicting Surface Exposed Regions

Surface exposed regions of Glu-C digested CPG2 peptides were determinedusing the X-ray crystal determined structure of CPG2 (Swiss-Prot. Pdbreference 1CG2), Insight II (MSI, UK) and the DSSP solvent accessibilityalgorithm (Rowsell, S. et al. Structure 5, 337-347 (1997)). Glu-Cgenerated fragments of CPG2 were identified on the Protein AnalysisWorksheet (PAWS) (ProteoMetrics, USA). Analysis of the CPG2 crystalderived conformational structure (Rowsell, S. et al. Structure 5,337-347 (1997)) showed that peptide Y[159-176]E was solvent exposed(Kabsch, W. & Sander, C. Biopolymers 22, 2577-2637 (1983))(solventexposure>78%) about residues Y[159-163]D, which we termed Region I,shown in FIG. 3 a. Peptide Y[391-415]K was solvent exposed aboutresidues 413-414, this was termed Region II (FIG. 3A). These solventexposed regions are only 6 Å apart between Asp163 of Region I and Ala413of Region II. The close spatial proximity of the two surface exposedregions supported their role as the CM79 binding epitope. Moleculardynamics modelling confirmed these results and moreover suggested that,with respect to region I (residues 159-163), residues 157 and 158 arealso surface exposed and therefore form part of the epitope. Similarly,molecular dynamics modelling suggested that in addition to residues 413and 414 of region II, residues 412 and 415 are also surface exposed andso may form part of the epitope. Indeed, as described below and as shownin Table 1, substitution of an alanine residue at residue 412 resultedin decreased CM79 binding. We hypothesized that if Regions I and IIcomprised a clinically relevant epitope that we had defined with CM79,then mutations of that epitope would reduce recognition by CM79 andpatients' antibodies.

SELDI-AMS combined with Glu-C proteolytic epitope mapping of theCPG2/CM79 complex identified five CM79 binding peptides derived fromCPG2 (FIG. 2). CPG2 fragments identified were numbered according to theCPG2 Swiss-Prot entry (accession code P06621) (FIG. 3 b). The observedpeptide masses were, 6269.8+H Da assigned to CPG2 R[356-415]K mass6270.2 Da (nearest alternative A[314-375]E mass 6281.1 Da) (FIG. 2A),4354.1+H Da assigned to CPG2 S[376-415]K mass 4354.1 Da (nearestalternative A[314-356]E mass 4365.0 Da) (FIG. 2A), 2794.9+H Da assignedto CPG2 Y[391-415]K mass 2794.4 Da (nearest alternative A[347-375]E mass2748.0 Da) (FIG. 2B), 3539.9+H assigned to CPG2 Y[159-189]E mass 3539.8Da (nearest alternative K[208-243]E mass 3537.0 Da) (FIG. 2B), and2092.8+H Da assigned to CPG2 Y[159-176]E mass 2092.2 Da (nearestalternative E[355-375]E mass 2063.2 Da) (FIG. 2C). The proteolyticfragments were all derived from two sequentially remote regions of CPG2.These results were obtained in duplicate on each assay chip and onsubsequent repetitions of the experiment. Control spectra (right side ofFIG. 2) were performed using non-CPG2 binding sFv M009.

1.3 Generation of MFE-23::CPG2-his Variants.

MFE-23, a recombinant scFv produced by filamentous phage technology, hasshown good localisation to CEA-producing tumours in patients. MFE-23 iswell characterised, is produced in high yields and has high affinity andspecificity for CEA. A radiolabeled fusion protein of MFE-23::CPG2expressed in E.coli has been shown to localise to colorectal tumourxenografts in nude mice(Bhatia et al, Int. J. Cancer 85 571-577, 2000).

MFE-23::CPG2 Fusion proteins were produced with variant CPG2. Changes to(KEYGVRD)(SEQ ID NO:2) were made by insertion of annealed pairs ofoligonucleotides encoding the substituted amino acids into HindIII andKpnI cohesive termini previously created by PCR mutagenesis (Purdy, D.et al. J. Medical Microbiol. 49,473-479 (1999)) to flank Region I inwild type (wt) CPG2 plasmid. Changes to (GAGK)(SEQ ID NO:4) were alsoprepared. Eight variants are illustrated in Table 1 and are labelled asV1 to V6.

CM79 binding and CPG2 enzyme activity of all CPG2 variants wereinitially tested in clarified culture supernatants for CM79 binding. Theresults, shown in Table 1, demonstrate that Arg162 was critical for CM79binding (sFv binding to V6 was up to 99% less than binding to wtMFE-23::CPG2, FIG. 4(wt=wild-type)). The variant V8 MFE-23::CPG2indicated the role of the c-terminus in CM79 binding of CPG2 (FIG. 4,Table 1). CM79 bound to all the remaining CPG2 variants (Table 1). TABLE1 CPG2 variants tested for CM79 binding and enzyme activity. A singlemutation was made in each variant (bold) in either the KEYGVRD or theGAGK regions. Wild-type MFE-23::CPG2 and variant V6 were CEA affinitychromotography and FPLC purified for CM79 binding, serum binding andenzyme activity assays. epitope Conformational Region 2 Enzyme CloneRegion 1 (C-term.) Activity CM79 binding wild-type KEYGVRD GAGK Yes YesCPG2 V1 AEYGVRD GAGK Yes Yes V2 KAYGVRD GAGK Yes Yes V3 KEAGVRD GAGK YesYes V4 KEYAVRD GAGK Yes Yes V5 KEYGARD GAGK Yes Yes V6 KEYGVAD GAGK YesReduced (up to 99%) V7 KEYGVRA GAGK None Yes V8 KEYGVRD AAGK Yes Reduced(up to 89%)1.4 ADEPT Patient Sera Binding to Variant V6 and Wild-type (wt) CPG2

As variant V6 was shown to have negligible binding to CM79 it wasselected for testing for reactivity with patient sera.

V6 and wild-type (wt) MFE-23::CPG2 were expressed and purified from6×250 ml culture volumes. Protein yields were 1.3 mg/l for V6 and 0.84mg/l for wt MFE-23::CPG2. Both proteins were radiolabelled using thechloramine-T method (Greenwood, F. C. & Hunter, W. M. Biochem. J. 89,116-123 (1963)). with 37 MBq iodine-125 sodium iodide added to 1 ml of0.5 mg/ml protein. Radiolabelled proteins were FPLC purified using aSuperose 12 column (Pharmacia; PBS mobile phase 0.5 ml/min). Gammaradiation emitting fractions at 135 kDa elution point were tested forCPG2 enzyme activity. Protein concentrations were determined using Lowryreagent according to the manufacturer's protocol (BioRad).

V6 was presented to 15 serum samples from patients who had antibodies toCPG2 as a result of receiving ADEPT. 2% BSA blocking, wash andO-phenylenediamine dihydrochloride (Sigma) detection stages were asdescribed by Bhatia et al(Int. J. Cancer 85 571-577, 2000). ELISA plates(Maxisorb, Nalgene-Nunc International, UK) were coated overnight at 4°C. with 0.5 mg CPG2, 0.02 mg V6 or 0.02 mg wt MFE-23::CPG2 per well.Library derived sFv binding to CPG2 was detected with mouse anti-myc tagantibody 9E10, followed by sheep anti-mouse IgG peroxidase conjugatedantibody (SAM-HRP) (Amersham Life Sciences, UK). To monitor CM79-Hisbinding to CPG2 or the MFE-23::CPG2 variants CM79 was detected usingmouse anti-His tag antibody (Dianova, Germany), followed by SAM-HRP. Thepatient serum inhibition assays were performed by saturating CPG2 coatedwells with CM79 at 25 mg/ml. ADEPT patient serum binding to CPG2 or theMFE-23::CPG2 variants was detected with goat anti-human IgG HRP (Sigma).

Results showed that all these sera had lower binding to V6 than the wtMFE-23::CPG2 (FIG. 5, Table 2). Extrapolating the maximum ELISA opticaldensity signal for undiluted serum, the antibody binding reductions inthe patient group had the range 10.2-65.3%, with a median reduction of45.1%. Rabbit anti-MFE-23 serum was used to detect the MFE-23 domains ofwt and V6 fusion proteins. As each CPG2 variant is linked to an MFE-23sFv, equimolar concentrations of the fusion proteins should haveequivalent anti-MFE-23 responses. The anti-MFE-23 binding responses forthe two fusion proteins varied by only 5.8%, a difference within thelimits of ELISA experimental error. TABLE 2 Percentage reduction of 15ADEPT patient sera binding to V6 compared to wt MFE-23::CPG2 determinedby ELISA. Rabbit anti-MFE serum is directed against the antibody domainof the fusion proteins and acts as a negative control. Decrease in Serumantibody binding (%) A1  30.1 A3  65.3 A4  13.3 A8  45.1 A11 62.0 A1248.7 A13 57.6 A14 56.0 A15 25.5 A16 56.7 A17 20.1 A18 10.2 A19 51.5 A2014.9 A21 30.2 Rabbit 5.8 anti-MFE

Example 2 Glycosylation of CPG Improves Retention of Fusion Proteins inTumours Despite Rapid Clearance from Normal Tumours

Although radiolabeled fusion protein of MFE-23::CPG2 expressed in E.colihas been shown to be promising candidate for ADEPT if favorable enzymedelivery is established (Bhatia et al, Int. J. Cancer 85 571-577, 2000),the yields obtained with this bacterially expressed product were too lowfor developing a clinical product. Therefore, the present inventors haveinvestigated expression in yeast Pichia pastoris. The Pichia pastorisexpressed product was constructed with tags for identification andexpression. Experiments performed with the Pichia pastoris expressedfusion proteins led to two unexpected findings:

-   a) that the glycosylated fusion proteins were retained in active    form in the tumour, despite rapid clearance from normal tissues    (described in the present Example);and-   b) that the presence of C-terminal tags reduced immunogenicity in    animal models (described in Example 3).    2.1 Glycosylation

Oligosaccharides were identified by collision induced disassociationmass spectrometry (CID) of trypsinated fragments of MFE-23::CPG2 gly-his(Pichia pastoris expressed MFE-23::CPG2 fusion protein with a C-terminalhexahistidine tag).

There are three potential N-glycosylation sites on CPG2, CID showed thatonly two of the three sites are glycosylated by Pichia pastoris.Asn222—Glycosylated, mostly mannose 5-13 chains Asn 264—Not glycosylated(not solvent exposed) Asn 272—Glycosylated, mostly mannose 8-10 chainsSome O-linked glycosylation (a maximum of five mannose) is present onthe MFE moiety of MFE-23::CPG2gly-his.

2.2 Pharmacokinetics & Biodistribution of MFE::CPG2gly-his in Mice

Nude mice bearing LS174T xenografts were injected i.v. with the fusionprotein (25 units per mouse). Blood samples were taken at differenttimes after injection. Plasma and tissues were assayed for CPG2 activityas follows:—CPG2 activity in tumor and normal tissues was measured byin-vitro turnover of the substrate methotrexate (MTX) by localisedenzyme and measurement of the metabolite peak by HPLC. Briefly, acalibration curve for each tissue was constructed by incubating therelevant tissue (taken from untreated mouse) with varying concentrationsof CPG2 and a fixed concentration of methotrexate and analysing thesolution by HPLC to give a standard line for CPG2 concentration v.metabolite peak area formed. Tissue homogenates were prepared in assaybuffer (PBS+Zinc Chloride) w/v 20% and diluted further as appropriateand incubated with methotrexate. The reaction was stopped by addition ofice cold methanol (1:1). The supernatant was analysed by HPLC.

2.3 Clearance

Results for plasma clearance of MFE-CPG2gly-his in comparison to thoseobtained with a chemical conjugate of monoclonal anti-CEA with CPG2(A5CP) are shown in FIG. 6. Rapid clearance of MFE::CPG2gly-his isdemonstrated.

2.4 Biodistribution of MFE-CPG2gly-his

To assess CPG2 activity in enzyme activity in LS174T xenografted nudemice, tumour, liver. kidney, lung and spleen were collected from 4 miceper time point at 2, 4 and 6 hours after intravenous injection ofMFE-23::CPG2 gly-his fusion protein and enzyme activity was assayed intissue extracts. Results, shown in FIG. 7, indicate that, despite rapidplasma clearance, enzyme activity persists in the tumours. In addition,selective localisation in the tumour occurs at much earlier time pointsthan observed with the A5B7-F(ab′)2-CPG2 conjugate. Rapid plasmaclearance in conjunction with high levels of CPG2 activity retention intumours resulted in tumour to plasma ratios of 163:1 at 6 hrs afterfusion protein injection. Tumour to liver, kidney, lung and spleenratios at 6 hrs post injection were 254,245, 158 and 160respectively(FIG. 8).

2.5 MFE-23::CPG2gly-his and MFE-23::CPG2gly-myc-his Remain Intact inTumour.

In addition to HPLC analysis of CPG2 enzyme activity in excised tissue(FIG. 7), the integrity of the enzymatically active material was testedto demonstrate that enzyme activity was due to MFE-23::CPG2 which hadremained intact in the tumour.

¹²⁵I radiolabelled MFE-23::CPG2gly-his or MFE-23::CPG2gly-myc-his wasinjected intravenously to nude mice-bearing LS 174T human colorectaltumour xenografts. Four hours later, the mice were sacrificed and thetumours were removed, homogenised and subjected to SDS-PAGE.Radiolabelled material was visualised by autoradiography. Thisdemonstrated that ¹²⁵I-MFE-23::CPG2 gly in the tumour had the samemolecular weight profile as ¹²⁵I-MFE-23::CPG2 gly prior to injection,indicating stability in vivo.

2.6 Relative Stability in Vivo

Effective targeted therapy relies on efficient antibody retention intumour after clearance from normal tissue and is primarily influenced bythe molecular size, stability and functional affinity of the antibody.Molecular size determines the circulating half-life of the antibody and,due to the dynamic recirculation between blood and tumour, ultimatelycontrols the uptake and residence time in tumour. Large antibodies showbetter tumour uptake and can deliver prolonged therapy but areimpractical due to their long circulating half-life. By contrast, smallantibodies show the perfect clearance pattern but are not retained intumour long enough to allow effective therapy. Ideally, the therapeuticwindow would be extended by improving stability and affinity. However,due to the obvious influence of molecular size it is difficult to assessthe isolated role of stability and affinity on retention. One way ofassessing retention, that is not related to molecular size is to comparethe area under the time-uptake curve for the tumour-specific antibodywith that of a nonspecific antibody that has the same characteristicclearance from blood. We have done this by fitting a bi-exponentialfunction to the blood data from the specific antibody. This function maythen be used along with a simple mathematical model, that assumesrecirculation between blood and tumour is caused only by a concentrationgradient between them, to simulate the equivalent time-uptake curve fora non-specific antibody. The area under each curve is obtained byintegrating to infinity with respect to time and the retention may beassessed by ratio of this integral for the specific relative to thenonspecific antibody. This ratio is 64.5 for the glycosylated fusionprotein (FIG. 9A) compared to only 4.4 for the non-glycosylated fusionprotein (FIG. 9B). Therefore, we estimate a potential 15-fold increasein therapeutic efficacy with the glycosylated product.

2.7 Therapy Studies Show Efficacy With MFE-23::CPG2gly-his andMFE-23::CPG2gly-myc-his Fusion Proteins

Mice were injected with fusion protein (25 units per mouse) i.v. whenthe tumours reached 0.1-0.2 cm³ and were in exponential growth. Theprodrug was given ×3 over 2 hrs between 4 and 6 hours after fusionprotein injection at 90 mg/kg per mouse i. p. Different control groupsincluded fusion protein alone (25 units/mouse), prodrug alone (90mg/kg×3) and a no treatment control group. Tumours were measured on dayone prior to treatment and subsequently on 3^(rd) or 4^(th) day untiltumour volume reached 2 cm³. The measurements were carried out in threedimensions (length, width and height) and the tumour volume estimated aslength×width×height divided by 2. The mean tumour volume (+/−sem) isplotted against days after treatment. The fusion proteins in combinationwith bis-iodo prodrug showed good growth delay of the tumour (see FIGS.10A & 10B).

2.8 Toxicity Studies

Mice were weighed prior to treatment and then ×2 weekly. Weight %relative to day 1 was calculated for each group of mice. Minimaltoxicity was observed, see FIG. 11A for MFE-23::CPG2gly-myc-his and FIG.11B for MFE-23::CPG2 gly-his.

Example 3 Tagging the CPG2 Protein Reduces Immunogenicity

This example demonstrates that tagging of CPG2 with his or myc-hisresults in reduced immunogenicity. In particular, MFE-23::CPG2gly-myc-his has very low immunogenicity.

3.1 Immunogenicity Assay

Balb/C mice (6-8 week old females) were injected with the followingfusion proteins (50 ug protein per mouse i.p) at one month intervals.

1. MFE-23::CPG2 gly-myc-his (P. pastoris)

2. MFE-23::CPG2gly-his (P. pastoris)

3. MFE-23::CPG2-his (E. coli)

4. MFE-23::CPG2 (E. coli).

Blood was collected from mice at 14 days post each immunisation. Bloodswere tested for mouse anti-CPG2 antibodies by standard ELISA procedures.Briefly, 96-well microtitre plates were coated with 100 μl of CPG2 (10μg/ml in coating buffer) overnight at 4° C. Wells were blocked with 250μl of 3% BSA for 1 hour and washed with PBS followed by distilled water.Mouse sera (×100 dilution) were incubated in duplicate for 1 hour at RT.Wells were washed with PBS/Tween followed by distilled water andincubated with sheep anti-mouse peroxidase antibody (10 μl/well at1/500dilution) for one hour. After washing, wells were incubated withsubstrate (100 μl /well) for 5 minutes for colour to develop. Thereaction was stopped by addition of 4M HCl. The plate was read atoptical density (O.D.)490 nm. SB43gal, a mouse monoclonal anti-CPG2antibody, was used as a positive control and normal mouse serum was usedas a negative control for ELISA reactions. O.D.'s were analysedstatistically using a non-parametric test (Mann-Whitney U-test). Resultsare shown in table 3. (O.D.—optical density, Fp—MFE-23::CPG2 fusionprotein, positive control—SB43gal, negative control—normal mouse serum).Results are shown for 10 mice for each fusion protein.

1. After the first immunisation there was no detectable immune responseto any of the MFE-23::CPG2 fusion proteins.

2. After the second immunisation there was a detectable immune responseto the E.coli expressed MFE-23::CPG2 with no tag (no.4 above) but not toany of the other constructs (nos 1-3 above). This difference wassignificant (p<0.001).

3. After the third immunisation there was a strong response toMFE-23::CPG2 with no tag, a weaker response to the -his taggedMFE-23::CPG2s (P. pastoris and E coli being similar) and a very lowresponse to the -myc-his tagged MFE-23::CPG2 (see table 3 for details).The differences were significant (p<0.001).

4. After the fourth immunisation there were stronger responses to the-his tagged MFE-23::CPG2s (P. pastoris>E .coli) but still a very lowresponse to the -myc-his tagged MFE-23::CPG2 (see table 3 for details).The difference between the -myc-his tagged fusion protein and the notagged fusion protein was significant (p<0.001). TABLE 3 Plate coatedwith CPG2 Mouse anti-CPG2 response after 1st immunisation Neg- Fpgly-Positive ative myc-his Fpgly-his e. coli-his e. coli no tag O.D. 0.4680.05 −0.1 −0.103 0.02 −0.069 −0.066 −0.095 −0.129 −0.098 −0.068 −0.091−0.076 −0.046 −0.07 −0.08 −0.087 −0.071 −0.058 −0.038 0.022 −0.031−0.091 −0.077 −0.073 −0.068 −0.094 −0.081 −0.044 0.017 −0.083 −0.0880.019 −0.062 −0.041 −0.104 −0.052 −0.098 −0.054 −0.05 −0.004 −0.103Mouse anti-CPG2 response after 2nd immunisation. Neg- Fpgly- Positiveative myc-his Fpgly-his e. coli-his e. coli no tag O.D. 0.519 0.0240.011 0.071 0.002 0.277 −0.033 −0.034 −0.026 0.308 −0.055 −0.035 0.0110.198 0.033 −0.012 0.024 0.092 −0.017 0.05 −0.025 0.228 −0.012 −0.0280.083 0.315 −0.01 −0.056 −0.009 0.232 −0.03 −0.041 0.078 0.313 −0.003−0.043 0.023 −0.015 −0.023 −0.027 0.034 0.116 Mouse anti-CPG2 responseafter 3rd immunisation Fpgly- positive negative myc-his Fpgly-his e.coli-his e. coli no tag O.D. 1.167 0.02 0.019 0.897 0.801 1.709 0.2220.514 1.4 1.618 0.028 0.095 0.794 1.374 0.005 0.98 1.436 1.289 0.0151.67 1.19 1.904 0.02 0.454 1.344 1.722 0.029 0.984 0.952 1.884 0.0210.791 1.202 1.775 0.039 0.55 0.86 1.077 0.025 1.416 1.523 1.454 Mouseanti-CPG2 response after 4th immunisation Fpgly- positive negativemyc-his Fpgly-his e. coli-his e. coli-no tag O.D. 0.91 0.005 0.037 0.5651.156 1.293 0.738 0.691 1.064 1.316 0.058 0.198 1.14 1.188 0.038 1.5911.14 0.765 0.09 1.182 0.934 1.208 0.244 1.463 1.011 1.415 0.13 1.5091.17 1.484 0.314 0.931 0.9 1.116 0.083 1.235 0.412 1.389 0.109 1.2020.693 1.198Negative numbers are below detection limits.Imune response to E. coli and P. pastoris produced MFE-23::CPG2 withdifferent tags

In conclusion, there is a difference in immunogenicity between thetagged and non-tagged fusion proteins; -his tag being slightly effectivein reducing the immune response and -myc-his tag being very effective.

The reduction in immunogenicity is apparently not due to glycosylationas there is no significant difference between the immune response toMFE-23::CPG2-his tagged fusion proteins from E.coli (not glycosylated)and P. pastoris (glycosylated).

3.2 Molecular Dynamic Modelling

Molecular dynamic modelling was used to simulate the interaction ofHis-tags and Myc-tags with the CPG2 molecule. The results are shown inFIGS. 12, 13 and 14. FIG. 12 shows that a charged histidine residue inthe hexa-His tag may interact with a residue (residue 163) from theepitope KEYGVRD(residues 157-163). Such interaction would explain thereduced immunogenicity seen when such tags are used. FIG. 13 shows thatan uncharged histidine residue in the hexa-His tag may interact with aresidue (residue K339) from the epitope EGGKKLVDK(residues 331-339).FIG. 14 shows that an a glutamate residue in the Myc tag may interactwith a residue (residue K339) from the epitope EGGKKLVDK(residues331-339), The modelling supports the observation that the tags reduceimmunogenicity.

1-15. (Canceled)
 16. An isolated carboxypeptidease enzyme, CPG2, inwhich an immunogenic region is modified to reduce immunogenicity to amammalian immune system whilst retaining CPG2 activity, wherein theimmunogenic region is selected from the group consisting of: (i)KIDGRGGK (SEQ ID NO.1) comprising residues 98-105 of SEQ ID NO.7; (ii)KEYGVRD (SEQ ID NO.2) comprising residues 157-163 of SEQ ID NO.7; (iii)YGVRD (SEQ ID NO.6) comprising residues 159-163 of SEQ ID NO.7; (iv)KLADY (SEQ ID NO.3) comprising residues 191-195 of SEQ ID NO.7; (v) GAGK(SEQ ID NO.4) comprising residues 412 to the C-terminal residue 415 ofSEQ ID NO.7; (vi) AG comprising residues 413-414 of SEQ ID NO.7; and(vii) EGGKKLVDK (SEQ ID NO.5) comprising residues 331-339 of SEQ IDNO.7.


17. An isolated or purified Pseudomonas carboxypeptidase CPG2 enzymewherein the C-terminus of the enzyme comprises an extension selectedfrom the group consisting of a histidine tag, a myc tag and a myc-histag.
 18. An isolated carboxypeptidease enzyme, CPG2, in which animmunogenic region is modified to reduce immunogenicity to a mammalianimmune system whilst retaining CPG2 activity, wherein the immunogenicregion is selected from the group consisting of: (i) KIDGRGGK (SEQ IDNO.1) comprising residues 98-105 of SEQ ID NO.7; (ii) KEYGVRD (SEQ IDNO.2) comprising residues 157-163 of SEQ ID NO.7; (iii) YGVRD (SEQ IDNO.6) comprising residues 159-163 of SEQ ID NO. 7; (iv) KLADY (SEQ IDNO.3) comprising residues 191-195 of SEQ ID NO.7; (v) GAGK (SEQ ID NO.4)comprising residues 412 to the C-terminal residue 415 of SEQ ID NO.7;(vi) AG comprising residues 413-414 of SEQ ID NO.7; and (vii) EGGKKLVDK(SEQ ID NO.5) comprising residues 331-339 of SEQ ID NO.7; wherein theC-terminus of the enzyme comprises an extension selected from the groupconsisting of a histidine tag, a myc tag and a myc-his tag.
 19. Thecarboxypeptidase enzyme of claim 16 wherein said enzyme is fused to anantibody other than an anti-CEA antibody.
 20. The carboxypeptidaseenzyme of claim 17 wherein said enzyme is fused to an antibody otherthan an anti-CEA antibody.
 21. The carboxypeptidase enzyme of claim 18wherein said enzyme is fused to an antibody other than an anti-CEAantibody.
 22. A method of preparing a fusion protein comprising acarboxypeptidase CPG2 enzyme of claim 17 and an antibody other than aCEA-antibody, comprising expressing a DNA sequence encoding said fusionoperably linked to a promoter in a Pichia pastoris host cell, andrecovering said fusion protein therefrom.
 23. A method of preparing afusion protein comprising a carboxypeptidase CPG2 enzyme of claim 18 andan antibody other than a CEA-antibody, comprising expressing a DNAsequence encoding said fusion operably linked to a promoter in a Pichiapastoris host cell, and recovering said fusion protein therefrom.
 24. Akit comprising a first component which is a prodrug which can beconverted to a cytotoxic drug by a carboxypeptidase of claim 16 and, asa second component, said carboxypeptidase.
 25. A kit comprising a firstcomponent which is a prodrug which can be converted to a cytotoxic drugby a carboxypeptidase of claim 17 and, as a second component, saidcarboxypeptidase.
 26. A kit comprising a first component which is aprodrug which can be converted to a cytotoxic drug by a carboxypeptidaseof claim 18 and, as a second component, said carboxypeptidase.
 27. A kitcomprising a first component which is a prodrug which can be convertedto a cytotoxic drug by a carboxypeptidase of claim 19 and, as a secondcomponent, said carboxypeptidase.
 28. A kit comprising a first componentwhich is a prodrug which can be converted to a cytotoxic drug by acarboxypeptidase of claim 20 and, as a second component, saidcarboxypeptidase.
 29. A kit comprising a first component which is aprodrug which can be converted to a cytotoxic drug by a carboxypeptidaseof claim 21 and, as a second component, said carboxypeptidase.